Construction Of Regulatable Cancer Targeted Gutless Adenovirus Vector Carrying Anticancer Gene To Treat Malignant Tumor

Gene therapy is a technique1 for expressing extrinsic genes by vectors based on rationally designed to correct or replace the abnormal genes. It is often used on hereditary diseases which are difficult to treat by traditional medical methods. Compared with normal hereditary diseases, cancer has the specialty of much more abnormal genes, and the more complicated mechanism. So using gene therapy to treat cancer is a little more difficult, but cancer gene therapy is still an important way. The key point is how to design the rational vectors. Gutless or gutted adenovirus has replaced all the wild type adenovirus genes by "stuff sequence"except the inverted terminal repeat (ITR) and the package signal. Its package needs the "helper virus" protein, so it is also called HD-ad(helper virus depended adenovirus) vector. It has many merits compared with traditional adenovirus vectors such as: lower immunogenic, higher extrinsic gene capacity, and longer gene expression. In this article we constructed a regulatable cancer target gutless adenovirus vector using hTERT(human telomerase reverse transcriptase) promoter and RU486 regulate system. At first, we use this vector carrying reporter genes to transfect cells in vitro, and we found in cancer cells the expression of reporter gene can be hardly detected without RU486, and the expression was much higher if RU486 added. The expression was found to be associated with the concentration of RU486 and the length of the induction. Then we used the vector carrying the anticancer gene Trail to generate the package plasmid GL-Ad-trail by homologous recombination in Kcoli strain BJ5183, and rescused the recombinant gutless adenovirus by cotransfecting the 293Cre4 cells with helper virus. After amplification and purification, we got enough gutless adenovirus. We found the recombinant gutless adenovirus can activate Caspase-3 and Caspase-8 to cause the apoptosis of cancer cells with the induction of RU486, but have no effect on normal cells. This result showed that the recombinant gutless adenovirus GL-Ad-trail can...